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Journal of Clinical Oncology, Vol 20, Issue 15 (August), 2002: 3282-3292
© 2002 American Society for Clinical Oncology

Expression of Multidrug Resistance Genes MVP, MDR1, and MRP1 Determined Sequentially Before, During, and After Hyperthermic Isolated Limb Perfusion of Soft Tissue Sarcoma and Melanoma Patients

By Ulrike Stein, Karsten Jürchott, Matthias Schläfke, Peter Hohenberger

From the Division of Surgery and Surgical Oncology, Charité, Humboldt University, Campus Berlin-Buch, Robert Rössle Hospital and Tumor Institute, and Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Address reprint requests to Ulrike Stein, PhD, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Straße 10, 13092 Berlin, Germany; email: ustein{at}mdc-berlin.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: Isolated, hyperthermic limb perfusion (ILP) with recombinant human tumor necrosis factor alpha and melphalan is a highly effective treatment for advanced soft tissue sarcoma (STS) and locoregional metastatic malignant melanoma. Multidrug resistance (MDR)-associated genes are known to be inducible by heat and drugs; expression levels of the major vault protein (MVP), MDR1, and MDR-associated protein 1 (MRP1) were determined sequentially before, during, and after ILP of patients.

PATIENTS AND METHODS: Twenty-one STS or malignant melanoma patients were treated by ILP. Tumor tissue temperatures were recorded continuously and ranged from 33.4°C initially to peak values of 40.4°C during ILP. Serial true-cut biopsy specimens from tumor tissues were routinely microdissected. Expression analyses for MDR genes were performed by real-time reverse transcriptase polymerase chain reaction and immunohistochemistry.

RESULTS: In 83% of the patients, MVP expression was induced during hyperthermic ILP. MVP-mRNA inductions often paralleled the increase in temperature during ILP. Increased MVP protein expressions either were observed simultaneously with the MVP-mRNA induction or were delayed until after the induction at the transcriptional level. Inductions of MDR1 and MRP1 were observed in only 13% and 27% of the specimens analyzed. Temperatures and drugs applied preferentially led to an induction of MVP and were not sufficient to induce MDR1 and MRP1 in the majority of tumors.

CONCLUSION: This study is the first to analyze the expression of MDR-associated genes sequentially during ILP of patients and demonstrates that treatment might lead to increased levels of MVP, whereas enhanced levels of MDR1 and MRP1 remain rare events.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ISOLATED LIMB perfusion (ILP) is an established treatment for locally advanced soft tissue sarcoma (STS) and metastatic malignant melanoma confined to the limb. By this means, high-dose regional chemotherapy is combined with hyperthermia, and increased antitumor activity in the extracorporeal circuit is expected. Recombinant human tumor necrosis factor alpha (rhTNF-{alpha}) plus melphalan1 is currently regarded as the most efficient drug combination in this setting. Multicenter trials reported complete (CR) and partial response (PR) rates of up to 82% for STS2,3 and from 65% to 100% for malignant melanoma.4,5

In sarcoma patients, resection of the residual mass after up-front rhTNF-{alpha}/ILP enabled limb-sparing treatment in approximately 90% of the patients who were candidates for amputation. Even with long-term follow-up, local recurrence rates are low, and consequently rhTNF-{alpha}/ILP significantly contributes to the management of extremity sarcoma in various centers.

Beyond the administration of rhTNF-{alpha}, treatment efficacy is thought to be based on the supportive effect of hyperthermic conditions during which drugs are applied. Comparative data obtained in melanoma patients from different centers indicated CR rates increasing from 27% to 54% if ILP temperatures were increased from less than 40°C to more than 41.5°C.6,7 An effect of hyperthermia on the efficacy of rhTNF-{alpha} to induce tissue necrosis in sarcomas was observed, and a steep decline of tissue oxygen partial pressure after rhTNF-{alpha} application was described to be indicative.8

Cancer therapy–related factors such as drugs, heat, or cytokines may induce or enhance the multidrug resistance (MDR) phenotype, which is associated with an increase in adenosine triphosphate–binding cassette (ABC) transporters: the MDR gene 1 (MDR1), which encodes P-glycoprotein (PGP)9,10; the gene MRP1, which encodes the MDR-associated protein MRP111,12; or both. The resistance mechanism of MDR might be crucial with respect to chemotherapeutic protocols, and several articles have analyzed MDR1 expression in sarcomas in the context of clinical outcome.13-19 MDR1/PGP induction was reported for a variety of drugs/chemicals and radiation in in vitro systems, but exposure to increased temperatures (mostly approximately 42°C to 43°C) also led to enhanced MDR1 levels, whether they followed single or repeated heat treatments.20-24 MRP1 overexpression was reported for human sarcoma and melanoma cell lines and for human tumors.25,26 MRP1 induction was observed by exposure to cytostatics drugs, but also after incubation at 43°C.24

The lung resistance protein (LRP), originally isolated from a non–PGP-expressing, but multidrug-resistant, tumor cell line,27 was later identified as the major vault protein (MVP).28 Vaults are organelles involved in nucleocytoplasmic drug transport with respect to the defense against xenobiotics. High MVP expressions were found in normal tissues chronically exposed to xenobiotics.29,30 MVP expression was also detected in clinical specimens, and correlation with clinical outcome parameters has been described for sarcomas and melanomas.31-34 MVP induction was observed after treatment with chemicals and cytostatics, including melphalan,35-38 that play a crucial role in the rhTNF-{alpha}/melphalan ILP setting.39 In contrast, incubation with rhTNF-{alpha} either externally applied or after gene transduction led to MVP downregulation in human tumor cell lines.40 No reports are available that describe the effect of heat on MVP expression.

From this point of view, the MDR phenotype of the tumor might be decisive for the success and efficacy of ILP. Each of its components—cytostatic drugs, cytokines, and heat—harbors the potential to modulate MDR. This is the first study to analyze biopsy specimens taken sequentially during treatment to evaluate the risk of inducing or enhancing the expression of MDR genes during ILP of STS and melanoma patients. Gene expression levels of MDR1/PGP, MRP1, and MVP were quantitatively and qualitatively determined and were correlated with temperature parameters and with clinical outcome.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
We collected serial biopsy specimens from 21 patients (11 females and 10 males; mean age, 49.2 years; range, 11 to 75 years) undergoing ILP for locally advanced STS (n = 14) or recurrent, bulky malignant melanoma (n = 7; Table 1). The mean tumor size was 8.8 ± 5.4 cm (mean ± SD; range, 2 to 24 cm). Sarcoma grading according to Trojani et al41 was grade 1 in two, grade 2 in six, and grade 3 in five cases and was undifferentiated in another case.


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  		Table 1. Patient Characteristics
 
Isolation Perfusion
The major artery and vein of the limb were surgically exposed and cannulated, and extracorporeal circulation was established with a roller pump and heat exchanger (Stöckert Corp, Munich, Germany) as previously described.42 The perfusate temperature was 39°C to 41°C, and according to the study protocol, tissue temperature had to be 38°C before drugs were administered and was kept at less than 40.5°C during the perfusion time (90 minutes). The rhTNF-{alpha} (Boehringer Ingelheim Corp, Ingelheim, Germany) dose was 3 mg (upper limb) or 4 mg (lower limb). The dosage of melphalan (L-PAM; Glaxo Wellcome, Hamburg, Germany) was 10 mg/L of the perfused limb volume measured with the water displacement method.43 Cisplatin (Medac, Hamburg, Germany) was administered at 0.5 to 0.8 mg/kg body weight, and doxorubicin (Adriamycin; Pharmacia, Erlangen, Germany) was administered at a dose of 15 to 30 mg. Leakage monitoring to the systemic circulation was performed by indium-111–labeled autologous RBCs and technetium-99m–labeled albumin.44 The mean leakage rate was 2.7% ± 5.2% of the activity applied. After drug perfusion, the limb was rinsed with 2 to 3 L of hydroxyethyl starch until no further reduction of the activity of the radiotracer could be achieved; vessels were decannulated and sutured, and the patients were transferred to the intensive care unit with cardiopulmonary monitoring for at least 24 hours.

The temperature within the tumor tissue, as well as within uninvolved muscles, was continuously recorded by polarographic electrodes (LICOX C8; GMS, Kiel, Germany). The median basal tumor temperature was 35.7°C (range, 32.2°C to 35.8°C), and the median temperature increase was 5.5°C (range, 1.3°C to 7.1°C). Tmax described the maximum temperature reached during ILP. ILP with rhTNF-{alpha}/melphalan followed the protocol of a multi-institutional trial3 that had been approved by the local ethics committee (Arbeitsausschuss 2, Humboldt University of Berlin, Germany). All patients gave their written informed consent.

Tissue Samples
Seven sequential tumor tissue biopsy specimens were obtained by using core needles (14-gauge; U.S. Biopsy, Franklin, IN). These were snap-frozen immediately and stored in liquid nitrogen at the following time points: before any manipulation, after induction of the extracorporeal circuit, when a 38°C tissue temperature was reached, 45 minutes after drug administration, after completion of the washout procedure, at the end of the operation, and at days 1 to 3 and 6 to 7 after ILP (Fig 1). Tissue samples were coded and blinded for expression analysis; decoding was performed after completion of all laboratory work.



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  		Fig 1. Procedure of hyperthermic isolated limb perfusion. Time points when biopsy specimens were taken are indicated by consecutive numbering. TNF, tumor necrosis factor; OP, operation.

 
Assessment of Tumor Response
Clinical response to limb perfusion. Clinical tumor response was assessed according to World Health Organization criteria45 by magnetic resonance imaging or computed tomography scans 4 weeks after surgery. As part of the evaluation of the study results of the rhTNF-{alpha}/melphalan protocol, indications for treatment and responses twice underwent external auditing.

Histopathology of the resection specimens. Resection of the residual mass was performed a median period of 8 weeks after ILP when the inflammatory reaction of the limb had resolved. Beyond standard histologic procedures, resection specimens were assessed in particular for the proportion of vital tumor areas and necrosis.46 A CR was defined as complete necrosis of the lesion with no vital tumor cells detected. To qualify for a pathologic partial remission, necrotic areas of at least 90% of the tumor had to be present. If less than 90% necrosis was found, a pathologic PR was accepted only if the criteria for clinical or radiologic PR were met.

Microdissection and RNA Isolation
Serial cryosections of each tissue were made for RNA isolation (10 µm) and immunohistochemistry (5 µm). For microdissection of tumor cell populations, each fifth cryosection per tissue was evaluated by a pathologist after staining with hemalum. Isolation of total RNA was performed from microdissected cell populations, including a DNAse incubation step (High Pure RNA Isolation Kit; Roche Diagnostics GmbH, Mannheim, Germany). RNA concentrations were measured in a microplate reader (RiboGreen RNA Quantitation Kit; Molecular Probes via MoBiTec, Göttingen, Germany) and were calculated in duplicate from the ribosomal RNA-standard curves (EasySoftG200/Easy-Fit software; SLT-Labinstruments, Crailsheim, Germany).

Relative Quantitative Two-Step Real-Time Reverse Transcriptase Polymerase Chain Reaction
Reverse transcriptase (RT) reaction was performed with 25 ng of total RNA (MuLV Reverse Transcriptase; Perkin Elmer, Weiterstadt, Germany). For each quantitative real-time polymerase chain reaction (PCR; 95°C for 30 seconds and 45 cycles of 95°C for 10 seconds, 62°C for 10 seconds, and 72°C 10 seconds), one fifth of the RT volume was taken by using the LightCycler DNA Master Hybridization Probes Kit (Roche Diagnostics). Expression of MVP and of the housekeeping gene glucose-6-phosphate dehydrogenase (G6PDH) was determined in duplicate from the same RT reaction. For MVP, a 114-base pair (bp) amplicon, and for G6PDH, a 113-bp amplicon were produced by intron-spanning primers, which were detected by gene-specific fluorescein- and LCRed640-labeled hybridization probes (syntheses of primers and probes: BioTeZ, Berlin, Germany; sequences of primers and probes: Roche Diagnostics). The calibrator cDNA was used in serial dilutions (in duplicate) simultaneously in each run (Fig 2), deriving from the doxorubicin-selected and MVP-overexpressing tumor cell line GLC-4/ADR (obtained from Rik J. Scheper, Free University Amsterdam, the Netherlands). MVP overexpression in GLC-4/ADR versus GLC-4 was proven by RT-PCR (LightCycler RNA Amplification Kit; Roche Diagnostics) and by immunoflow cytometry using monoclonal MVP-specific antibodies before these experiments. Relative quantification of MVP expression in the tissue samples was performed as follows:



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  		Fig 2. Expression of MVP (A) and G6PDH (B) in GLC-4/ADR cells. RNA concentrations were 50, 20, 10, 5, 2, 1, and 0.5 ng; cDNA was used from the same RT reaction for MVP and G6PDH (in duplicate).

 
equation


For detection of MDR1 and MRP1 transcripts, real time RT-PCR was performed with MDR1- and MRP1-specific primers that amplified a 167-bp product for MDR147 and a 291-bp product for MRP148 (LightCycler RNA Amplification Kit). Copy number was determined by serial dilutions of transcripts for MDR1 and MRP1 (105 to 108 copies). Total RNA isolated from MRP1- or MDR1-overexpressing drug-selected calibrator tumor cell lines was simultaneously used: KBV-1 cells for MDR1 (provided by M.M. Gottesman, National Cancer Institute, Bethesda, MD) and MCF-7/VP16 cells for MRP1 (obtained from E. Schneider, Wadsworth Center, Albany, NY). These cell lines have been characterized before this analysis concerning their resistance gene expressions by use of RT-PCR, immunoflow cytometry, and functional assays.

Immunohistochemistry
Tissue sections from the same series as those taken for microdissection were used. Sections were fixed with acetone (10 minutes at -20°C), air-dried, and stored at -20°C. The specimens were stained with the alkaline phosphatase-antialkaline phosphatase (APAAP) dual system according to the recommendations of the manufacturer (Dianova, Hamburg, Germany). Briefly, sections were preincubated in Tris-buffered saline (50 mmol/L of Tris-HCl, 150 mmol/L of NaCl, pH 7.6), and treated with the primary antibody in RPMI dilution medium (9% RPMI, 9% inactivated bovine serum, and 0.9% sodium azide) for 60 minutes. The following monoclonal antibodies were used: LRP/LRP56 (1:50), MRP/MRP-m6 (1:50), PGP/JSB-1 (1:20), and C219 (1:20). After washing, incubation with a rabbit/anti-mouse bridge antibody (1:100; Dako, Glostrup, Denmark) was performed for 30 minutes. After additional washing, slides were treated with the APAAP dual system complex (1:80) for 30 minutes. The last four steps were repeated with incubation times of 15 minutes. After washing, slides were incubated in a New Fuchsin (Sigma, Taufkirchen, Germany) solution for 30 minutes. The nuclei were counterstained with hemalum. Slides were mounted with Mowiol (Hoechst, Frankfurt/Main, Germany).

Expression levels of MDR proteins were semiquantified by an immunoreactive score (range, 0 to 3) that measured staining intensity and the number of stained cells in each section. Slides stained without primary antibodies were used as controls. The specificity of the antibodies was established by Western blot and immunostaining with high- and low-expressing cell lines for each of the proteins.

Statistical Analysis
Data are presented as mean ± SD. Nonparametric tests according to Mann-Whitney and the Wilcoxon signed-rank tests were performed to compare independent and related samples. For correlation analysis between variables, Spearman’s correlation coefficient was calculated. Statistical analysis was performed with Excel/WinStat, Microsoft Office 6.0 (Microsoft, Redmond, WA), and SPSS for Windows, release 10.7 software (SPSS Inc, Chicago, IL).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of ABC Transporters MDR1/PGP and MRP1 During ILP
Expression of the ABC transporters MDR1/PGP and MRP1 was analyzed by real time RT-PCR and immunohistochemistry for the first 15 patients who were entered onto the study (patients no. 1 to 4, 6 to 9, 11, 12, 14, 15, 18, 19, and 21; Table 1). Basal MDR1 expression was determined in five of 10 sarcomas and in one of five melanomas. Induction of MDR1 expression levels was observed in two of 10 sarcomas (patients no. 1 and 8; 13% of the 15 tumors analyzed). Basal MRP1 expression was detected in nine of 15 tumors (five of 10 sarcomas and four of five melanomas), whereas increases in MRP1 levels during treatment were observed in four sarcomas (27%).

Modulation of MVP Expression During ILP
Expression of MVP mRNA was found to be increased in 15 (83%) of 18 patients during ILP when compared with the respective basal levels before operation (set 100%; Fig 3). Levels of MVP inductions varied from 133% to 600%. MVP expression was decreased during treatment in three patients to 79%, 81%, and 92% of pretreatment values.



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  		Fig 3. Expression of MVP during hyperthermic ILP in all patients. MVP expression of patient no. 18 was 600% induced (depicted separately for better resolution of other patients’ values). MVP modulation of patients no. 2, 4, and 14 could not be evaluated because of limited availability of biopsy specimens.

 
MVP Expression and Temperature During ILP
There was a positive correlation between the maximum MVP expression reached during ILP and the respective Tmax in the 14 STS (r = .35; P = .05) The direct correlation of MVP expression and temperature determined at the respective time point during rhTNF-{alpha} drug application for each patient resulted in a correlation coefficient of .51 (P = .039; Fig 4). No correlation could be found in melanoma patients.



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  		Fig 4. Correlation analysis of MVP expression with temperature during TNF drug application. MVP expression was determined versus G6PDH as the quotient with the MVP/G6PDH ratio of the calibrator RNA; r = .51; P = .039.

 
Individual Courses of MVP Expression During ILP
To illustrate, individual courses of intratumoral temperature and MVP expression during ILP are presented in Figs 5 and 6. For patient no. 1, real-time RT-PCR performed for MVP and G6PDH is presented in Figs 5A and 5B. Curves of both graphs were within the linear range of these genes when compared with the respective calibrator graphs (Fig 2). On the basis of this, MVP expression was calculated and depicted in the context of the temperature measured during ILP (Fig 5C). Hyperthermia was induced from 35.2°C before operation to 38.0°C before rhTNF-{alpha}/melphalan and was maintained at 38°C during perfusion. In parallel, MVP expression was increased from 0.55 before operation and 0.82 before rhTNF-{alpha}/melphalan application to 1.27 during treatment (228% increase). After washout, the temperature decreased to 34.9°C and the MVP expression value decreased to 1.17 and to 0.47 at the end of operation. Thus, changes in temperature during ILP were followed by changes in MVP expression levels. Expression of MVP by immunohistochemistry was found to be induced within sections obtained at the end of heating and before rhTNF-{alpha}/melphalan application (Fig 5E), during treatment (Fig 5F), and after washout (Fig 5G). In contrast, low expression was detectable at the beginning (Fig 5D) and at the end of operation (Fig 5H). The data for MVP protein expression mirrored the results of MVP expression obtained at the transcriptional level.



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  		Fig 5. MVP expression for patient no. 1. Real-time PCR for MVP (A) and G6PDH (B) in duplicate. (C) Relative MVP expression and intratumoral temperatures. (D-H) Immunohistochemistry: beginning of operation (D); before (E) and during (F) TNF drug; after washout (G); and at the end of operation (H).

 


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  		Fig 6. MVP expression for patient no. 17. Real-time PCR for MVP (A) and G6PDH (B) in duplicate; (C) relative MVP expression and intratumoral temperatures. (D-G) Immunohistochemistry: beginning of operation (D); during TNF drug (E); after washout (F); at the end of operation (G); and after 1 day (H).

 
For comparison, MVP expression and temperature data for patient no. 17 are depicted in Fig 6. MVP expression by RT-PCR (Fig 6A and 6B) was found to be increased from 0.97 at the beginning of operation to 1.5 during rhTNF-{alpha}/melphalan treatment (155%), paralleling the increase of temperature from 34.1°C to 39.2°C (Fig 6C). After washout, the temperature decreased to 38.2°C and was 36.5°C at the end of operation. MVP expression was also reduced after washout (1.12) and diminished further at the end of operation (1.07). Thus, changes in temperature were paralleled by changes in MVP expression. At the protein level, MVP expression was also induced (Fig 6D to 6H; this was most prominent at the end of operation [Fig 6G]) and was reduced to the pre-ILP level on day 1 after ILP (Fig 6D and 6H). Although MVP protein expression was increased during ILP, as in patient 1, the increase in mRNA expression level preceded the induced expression of the respective protein.

Correlation Analyses of MVP Expression With Necrosis and Clinical Response
To discover whether MVP expression might be predictive for parameters of the clinical outcome of hyperthermic ILP, we correlated MVP expression levels and histologic, as well as clinical, response. Two of three patients who developed a complete tumor regression had the lowest MVP inductions. Additionally, six of 10 patients classified as "no change" were within the highest MVP increment. However, a single patient (no. 18) developed a CR despite having the absolute maximum increment of MVP during ILP; this prevented statistical significance for a general analysis. If this patient were excluded, the correlation between MVP increment and response would be .35 (P = .06). There was no statistically significant influence of histologic subtypes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Since its introduction to clinical application in 1957,49 ILP has become a treatment option for regional metastasis of melanoma and locally advanced STS. In vitro observations of increased tumor cell killing under increased temperatures led to the concept of hyperthermic limb perfusion.50,51 The introduction of cytokines, particularly rhTNF-{alpha}, and immune-modulating substances52 has attracted the utmost attention during the recent years.1,53 Increasing the temperature to 39°C to 41°C can significantly improve tissue perfusion and the distribution of cytostatic agents. Although cytotoxic effects of hyperthermia itself can be demonstrated at more than 42°C, a synergism with melphalan and cisplatin was shown at lower temperatures.54,55

The induction of MDR genes by cancer therapy–related factors might limit the success of chemotherapy in patients. So far, statements about modulation of MDR genes by hyperthermia in patients were restricted exclusively to the time points before and after hyperthermia. MDR1 expression was found to remain almost unchanged in the majority of patients with locally advanced rectal carcinomas treated with radiochemothermotherapy when analyzed before and after treatment.56 Another report describing the immunohistochemical expression of MDR1, MRP1, and MVP in STS before and 6 weeks after rhTNF-{alpha}/ILP could not detect an induction of MDR.57 Also, in our study, the expression levels of these genes were compared before treatment and at the time point of resection (on average 8 weeks after hyperthermic ILP): increased MVP gene expressions were detected in the minority of the cases and were determined exclusively in samples obtained from patients who had an increased MVP expression during hyperthermic ILP. A delayed induction of MDR1 and MRP1 at the time point of resection was not found.

However, we demonstrated a fast induction of MVP during ILP in the majority of the analyzed tumors. Fast induction courses similar to those seen here were described for MDR1 in metastatic sarcomas after application of the MDR-related drug doxorubicin.16 However, MDR1 upregulation was not found in the majority of our ILP-treated tumors, reflecting that a Tmax of approximately 40°C was not sufficient to induce or increase MDR1 expression generally and that melphalan does not belong to the classic panel of MDR-relevant drugs that induce ABC transporter expression. From in vitro studies it is known that MDR1 and MRP1 expression can be induced by heat exceeding 40°C.20-24 However, in perfusion therapy, temperatures beyond 42°C are accompanied by severe toxic reactions that sometimes necessitate amputation.50,58-60

It might be hypothesized that a threshold temperature is required to induce signal transduction cascades that result in the induction of ABC transporters, in phosphorylation, or in membrane topology alterations of PGP.61,62 Heat shock–responsive elements have been identified within the MDR1 promoter63-66 but not within the MRP1 promoter.67 However, we demonstrated recently that the hyperthermia-induced binding of the transcription factor YB-1 to the Y-box of the MDR1 promoter or to GC-rich sequences of the MRP1 promoter led to an increase in MDR1/PGP or MRP1 levels at 43°C.24 Furthermore, temperature-dependent activity of the heat shock transcription factors 1 and 3 was also demonstrated.68

Within the MVP promoter, we identified several consensus elements, including Y-box, E-box, and the p53 binding site.69 Thus, ILP modalities such as heat, melphalan, or rhTNF-{alpha} might act via these elements to regulate MVP.35-37 MVP expression can be diminished by rhTNF-{alpha} in tumor cells,40 but this effect might be masked by the effect of heat in the ILP setting. Thus, MVP reduction after washout or at the end of operation could be due to decreased temperature, rhTNF-{alpha} application, or both. We observed persistently increased MVP expression during the first postoperative days (eg, patient no. 1, Fig 5C), an effect that might be due to the systemic inflammatory response syndrome’s resolving until day 4 to 5 after surgery.70-73

There was no correlation of MDR gene expression, either basally or induced by ILP, with histologic or clinical response. The overall MDR status does not predict the success of ILP, and it is interesting to note that MVP induction did not limit the clinical result. This is even more important, because the cytostatics used—melphalan and cisplatin—have already been associated with the MVP-relevant drug spectrum.38,74 Modulation of drug effects by hyperthermia is well established: increased drug accumulations after heat treatment were repeatedly observed that led to chemosensitization toward several drugs.23,75 Because we intended to obtain biopsy specimens from nonnecrotic areas, some patients, in whom we could not isolate RNA from all serial biopsy specimens, had to be excluded from analysis. We probably investigated a negatively selected group of patients who did not develop complete necrosis of the tumor. Thus, the percentage of necrosis in the resection specimen does not correlate with findings in immunohistochemistry and RT-PCR for MDR-associated genes.

The activity of rhTNF-{alpha} in ILP is thought to be based on the multiple actions of rhTNF-{alpha} on endothelial cells in the tumor vascular system, and only tumors with microvascularization can be destroyed by rhTNF-{alpha}.76 In experimental tumors in vivo, rhTNF-{alpha} induces hemorrhagic necrosis.77 Besides antiproliferative and cytotoxic activity,78 rhTNF-{alpha} induces morphologic changes, increased expression of cell-surface receptors and adhesion molecules, and procoagulant activity,79,80 but it has also been reported to cause increased drug concentrations.39,81,82

In conclusion, this is the first study to provide data about expression modulation of MDR genes determined sequentially before, during, and after hyperthermic ILP by using rhTNF-{alpha} and cytostatic drugs. Although induction of MVP was found in the majority of the tumors, there was no correlation with histologic or clinical response to therapy. Further studies are required to clarify whether locoregional hyperthermia treatment at higher temperatures might lead to the induction of MDR genes, including ABC transporters, and whether these gene inductions may complicate simultaneous or subsequent chemotherapy. Such information is required to assess the risk of an MDR-induced phenotype and could contribute to redesigning the sequence of chemotherapy and cytokines.


   ACKNOWLEDGMENTS
 
Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 273, hyperthermia, project C13) and by Roche Diagnostics GmbH, Penzberg, Germany.

We thank Christoph Kettelhack, MD, and Wolfgang Haensch, MD, Robert-Rössle-Hospital, Berlin, for clinical and histologic support. We are grateful to Ina Wendler, Lisa Bauer, and Lieselotte Malcherek for excellent technical assistance.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Lienard D, Ewalenko P, Delmotte JJ, et al: High-dose recombinant tumor necrosis factor alpha in combination with interferon gamma and melphalan in isolation perfusion of the limbs for melanoma and sarcoma. J Clin Oncol 10: 52-60, 1992[Abstract]

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3. Eggermont AMM, Schraffordt Koops H, Klausner JM, et al: Isolated limb perfusion with tumor necrosis factor and melphalan for limb salvage in 186 patients with locally advanced soft tissue extremity sarcomas. Ann Surg 224: 756-765, 1996[CrossRef][Medline]

4. Kettelhack C, Hohenberger P, Schlag PM: Die isolierte hypertherme Extremitätenperfusion beim malignen Melanom mit Melphalan und Tumornekrosefaktor. Dtsch Med Wschr 122: 177-181, 1997[Medline]

5. Lienard D, Eggermont AMM, Schraffordt Koops H, et al: Isolated perfusion of the limb with high-dose tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and melphalan for melanoma stage III: Results of a multi-centre pilot study. Melanoma Res 4: 21-26, 1994 (suppl 1)[Medline]

6. Di Filippo F, Calabro A, Giannarelli D, et al: Prognostic variables in recurrent limb melanoma treated with hyperthermic antiblastic perfusion. Cancer 63: 2551-2561, 1989[CrossRef][Medline]

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8. Hohenberger P, Bida B, Schlag PM: Alteration of tissue oxygen partial pressure during isolated, hyperthermic limb perfusion with cytostatic drugs or recombinant human tumor necrosis factor alpha combined with melphalan. Strahlenther Onkol 172: 214-215, 1996 (suppl 2)

9. Ueda K, Yoshida A, Amachi T: Recent progress in P-glycoprotein research. Anticancer Drug Des 14: 115-121, 1999[Medline]

10. Litman T, Druley TE, Stein WD, et al: From MDR to MXR: New understanding of multidrug resistance systems, their properties and clinical significance. Cell Mol Life Sci 58: 931-959, 2001[CrossRef][Medline]

11. Cole SPC, Deeley RG: Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP. Bioessays 20: 931-940, 1998[CrossRef][Medline]

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Submitted January 2, 2002; accepted April 20, 2002.




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